1. Field of the Invention
The present invention relates generally to the fields of pathobiology, mycology and immunology. More particularly, it concerns the design of an model animal system that is analogus to the development of human invasive pulmonary aspergillosis and, therefore, provides a useful in vivo tool for studying the pathogenesis of disseminating mold infection, as well for the development of novel antifungal agents. It also provides a tool to prospectively follow the effects of various novel therapeutic interventions against such infections.
2. Description of Related Art
Disseminated fungal infections constitute one of the most difficult challenges for clinicians caring for patients with hematological cancer (Anaissie et al., 1989). While the incidence of Hematogenous candida infections has been significantly reduced through the prophylactic use of azoles, such as fluconazole, infections with opportunistic molds are now a leading cause of infectious mortality in this patient population (Anaissie 1992). Aspergillosis clearly remains the most common mold infection in patients with hematological cancer, with Aspergillus fumigatus being the offending cause in more than 90% of the infected patients. However, new opportunistic pathogens also have emerged worldwide as causing life-threatening infection, the most frequently reported of which is Fusarium spp. (Morrison et al., 1993; Morrison et al., 1994; Pfaller et al., 1992; Blazar et al., 1984; Uzun et al., 1995). Infections with Fusarium are associated with a very high mortality risk, and this mold is typically refractory to Amphotericin B, the standard therapy for disseminated mold infections. Since infection with this organism may mimic aspergillosis, patients are usually treated with Amphotericin B (AMB), an agent with poor activity against fusariosis. Further, similar to the case of Aspergillus infection, the airways are the most common primary site of inoculation/infection and are almost always involved in disseminated disease (Morrison et al., 1993; Morrison et al., 1994; Pfaller et al., 1992; Blazar et al., 1984; Uzun et al., 1995).
It has recently been suggested that the addition of gamma-interferon and/or GM-CSF might enhance the efficacy of AMB against opportunistic mold infections. This is important, since AMB by itself has only borderline efficacy against molds. Further, AMB treatment for documented or suspected systemic mold infections carries with it common (in  greater than 75% of the cases), substantial and frequently dose-limiting nephrotoxicity, occasionally serious enough to warrant hemodialysis. The acute infusion-related adverse events (severe shaking chills, fever, nausea, vomiting, and headache) also are quite troublesome to patients. Less common side effects encountered with the use of AMB include cardiac arrhythmia, bone marrow suppression, neuropathies, and convulsions (Sande et al. 1985).
Mold infections continue to pose a serious threat to the recovery of immunosuppressed patients who have undergone chemotherapy or radiotherapy. Thus, in addition to finding new and more effective antifungal antibiotics, the need for an animal model of systemic aspergillosis has been deemed valuable by many investigators in the art to improve an understanding of the pathophysiology of mold infections. The literature contains a multitude of reports describing invasive aspergillosis in small animal""s, such as mice, rats, and rabbits (Epstein et al., 1967; Dixon et al., 1989; Hector et al., 1990; Eisensten et al., 1990; Nawada et al., 1996; Turner et al., 1976; Kurup et al., 1981; Spreadbury et al., 1989; Francis et al., 1994). These reports clearly demonstrate the principle that an Aspergillus infection can be established in an experimental animal. However, none of these described models can be claimed to be clinically relevant, since the animal""s typically have been exposed to extraordinarily high fungal loads, and in most instances have been immunosuppressed with steroids alone (Epstein et al.; 1967, Hector et al., 1990; Nawada et al.; 1996, Kurup et al., 1981). Further, in all described models known to the inventors of the present invention, the fungal load is either infused intravenously or spread through intranasal injection to the lungs through the trachea or directly injected into the trachea transcutaneously. This produces a diffuse pulmonary infection, which is very different from what is typically encountered in an immunocompromised human patient. Thus, there is no available animal model that has a close similarity to the human clinical situation.
To overcome these and other deficiencies in the art, the present inventors have developed a model which closely resembles the clinical situation where a human patient, who has undergone intensive chemotherapy or radiotherapy as treatment of a malignancy, or as preparation for hemopoietic stem cell transplantation, subsequently suffers a mold infection.
Thus, the model developed is analogous to the commonly observed human situation, where the infection starts with a localized lesion and then disseminates to involve a majority of the pulmonary tissue, leading to the animal""s demise. The infective lesion often manifests as development of pneumonia in the animal model and in humans.
The development of a clinically relevant model for immunocompromised patients with systemic mold infections permits:
acquisition of an increased understanding of the pathophysiology of systemic fungal infections, such as is warranted to design new and more effective strategies for the prevention and treatment of these infections in immunocompromised human patients;
obtaining a xe2x80x9ctesting vehiclexe2x80x9d for the preclinical screening of novel antifungal agents and also for testing the efficacy of combinations of interventions to control an already established infection. This is of importance as it avoids unnecessary and cumbersome clinical trials where patients might otherwise suffer suboptimal treatment for their infection;
development of new methods for early detection of mold infections; and
obtaining a model for the long-term follow-up of the treatment of an established systemic fungal infection.
In a preferred embodiment the mold infection is by Aspergillus spp. The animal used herein as a the model animal is a beagle.
An important feature of the invention is that the animal model developed herein is reproducible. Thus, in one embodiment, longitudinal and systematic studies on the pathophysiology of the infection may be performed using the model.
In one important embodiment, the model may be used to develop or test new antifungal agents in vivo. In a specific example, the inventors present evidence for the testing of a new antifungal agent, Natamycin, using the model. In another embodiment, the model can be used to develop effective antifungal therapies, such as the use of combination drugs in vivo. In yet another embodiment, this in vivo model may be used to tailor the antifungal therapy to the anticancer therapy that the patient would be undergoing. The model developed herein also may be used for the development of long-term follow-up treatments. Primarily, the inventors envision that the use of this model should help save patients from clinical trials of antifungal drugs that may be effective in vitro without living up to the expectations in a clinical setting.
In a related embodiment, the model developed in the present invention can be used to develop methods for the early detection of Aspergillus infection.
Thus, the invention describes the use of a beagle, which is accepted by both the NCI and the FDA for safety studies of pharmaceutically active agents prior to entering clinical trials, as a clinical model for an immunocompromised patient that contracts a systemic mold infection. In alternate embodiments, the inventors also contemplate the use of other large animal""s, such as dog, pig, sheep, monkey, or chimpanzee for the animal model.
In one preferred embodiment of the invention, immunosuppression in a beagle is achieved by total body irradiation (TBI). In one specific embodiment, the TBI is achieved using an X-ray (Cobalt) source. The X-ray therapy (XRT), can be followed with daily oral steroid administration to further suppress T-lymphocyte and macrophage function and enhance spreading the infection. In alternative embodiments, the use of immunosuppressant steroids prior to the TBI is also contemplated. One of the steroids that may be used effectively is prednisolone. However, one of skill in the art will recognize that the use of other immunosuppressant steroids, such as hydrocortisone, betamethazone, glucocorticoid analogs, and others is also possible. As an alternative to steroids, the use of other T-cell suppressive agents such as anti-T-lymphocyte globulin (ATG), and/or nucleoside analogs (e.g. fludarabine), and/or immunophilins (e.g. Cyclosporin A or rapamycin) also are contemplated. The infective agent used to infect the model animal is a mold species. In a specific embodiment, the mold species chosen to infect the beagles is any one of the Aspergillus spp. In a preferred embodiment of the invention the mold species chosen is Aspergillus fumigatus. Aspergillus fumigatus accounts for over 90% of the human clinical infections. In alternative embodiments, any Aspergillus spp. that cause human clinical infections may be used.
In another preferred embodiment of the invention, the beagle is infected by Aspergillus spores. In a related aspect of the invention, the infection with spores is repeated on more than one occasion. In another related embodiment, the Aspergillus spores are encapsulated in small agarose beads. Alternatively, the mold spores may be encapsulated in other formulations used for encapsulation purposes as are known to those of skill in the art. Aspergillus spores so encapsulated provide a standardized dosage and can be cryopreserved by techniques known in the art to obtain a stock of a reproducible dosage of infectious inoculum.
An important aspect of the invention relates to the administration of the infective mold in a manner as to obtain a localized infective lesion in the model animal which is similar to the initial infection developed in immunosuppressed human patients. Thus, the mold administration is performed herein using a pediatric bronchoscope to safely and reproducibly introduce the mold in the intermediary (lower) right lung lobe.
In one embodiment of the invention, the time of mold administration to the beagle is at the time of leukopenia. In another embodiment of the invention, the time of mold administration to the beagle is at the time of profound immunosuppression. In a related aspect, the time of mold administration to the beagle is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 days after TBI or more preferable 10-14 days after TBI. In other aspects, the time of mold administration is when the animal""s neutrophil count is about  less than 400 per xcexcL.
In other aspects, prophylactic oral antibiotics are administered to the immunosuppressed beagles to prevent the development of secondary bacterial infections. The antibiotics used can be broad-spectrum. In several cases, the antibiotic regimen is changed as deemed necessary to obtain better antibacterial coverage, while still allowing for the mold infection to progress. In related aspects of the invention, prophylactic platelet transfusions are administered to the immunosuppressed beagles used in the model system to prevent bleeding.
It is envisioned that this model system may be suitably adapted for studying a variety of mold infections, and developing novel antifingal drugs in immunocompromised patients such as patients undergoing BMT, chemotherapy, broad-spectrum antibiotics, cytotoxic therapy, immunosuppressants, patients with AIDS, or/and patients with intravascular catheters.